This report includes work arising from the following clinical protocols: NCT00005011, NCT00056901, NCT00059228, NCT00082043, NCT00100360, NCT00001177, NCT00001259, and NCT00001481. Our studies have documented that PMDD symptoms are eliminated by ovarian suppression and stimulated by administration of ovarian steroids yet appear in the context of levels of ovarian steroids indistinguishable from those in women without PMDD. Additionally, we demonstrated that the change in levels of estradiol and progesterone (E/P) from low to high, and not the steady state levels, was associated with the onset of PMDD symptoms. Our findings provided a new target on which interventions could be focused (namely increasing neurosteroid levels from the follicular to the luteal phase). To that end, we completed a pilot study evaluating (under blinded conditions) the effects of inhibiting the production of the neurosteroid, allopregnanolone (a derivative of progesterone that is implicated in the alterations of neurotransmitter function that could lead to PMDD). After successful elimination of the luteal phase increase in allopregnanolone (despite maintaining normal progesterone levels), the emergence of luteal phase affective symptoms in PMDD were prevented. These findings have led to our developing a new protocol to be submitted in 2019 in which we will conduct a larger placebo-controlled treatment trial investigating the effects of stabilizing neurosteroid levels with dutasteride in women with PMDD. We also employed our ovarian steroid manipulation studies (GnRH agonist studies) to explore possible neural substrates of risk in women with PMDD. We have identified the subgenual anterior cingulate cortex to be differentially regulated by ovarian steroids in women with PMDD compared with control women (i.e., regional cerebral blood flow rCBF during a resting state exam is decreased during estradiol or progesterone exposure when PMDD symptoms recur). The degree of altered rCBF also correlates with gene expression in the ESC/E(Z) pathway of genes a family of genes which we previously identified to be differentially expressed (increased) in cell lines from women with PMDD compared to controls, and members of which are differentially regulated by estradiol and progesterone in cell culture (see below). These data suggest both the relevance of differences in the response of the subgenual cingulate cortex to ovarian steroids with consequent differential activation of brain networks that mediate the affective responsivity of women with PMDD as well as providing a biologically plausible target for neuromodulatory interventions in PMDD. These behavioral and neuroimaging data also set the stage for the performance of in vitro cellular studies of ovarian steroid responsivity in women with PMDD. In collaboration with Dr. David Goldman at NIAAA, we developed lymphoblastoid cell lines (LCLs) and human induced-pluripotent cell lines (h-IPSCs) from women with and without PMDD who had participated in our GnRH agonist-induced ovarian suppression studies. The behavioral outcomes observed during these protocols serve to refine the hormone-sensitive phenotype beyond simply the established clinical diagnoses. Our in vitro experimental strategies attempt to recapitulate the endocrine events that trigger mood symptoms in women with PMDD.In this study, pathway analyses of the LCL transcriptome revealed, among others, over-expression of ESC/E(Z) complex genes (an ovarian steroid-regulated gene silencing complex) in untreated LCLs from women with PMDD, with more than half of these genes over-expressed as compared to controls. This pattern of increased ESC/E(Z) mRNA expression was confirmed in the larger replication cohort by qRT-PCR. In contrast, protein expression of ESC/E(Z) genes was decreased in untreated PMDD LCLs. Finally, mRNA expression of several ESC/E(Z) complex genes were increased by P in controls only and decreased by E in PMDD LCLs. These findings provided the first evidence of a plausible biological substrate for the differential behavioral response to E/P in women with PMDD. Indeed, these data suggest that women with PMDD have an intrinsic abnormality in their epigenetic capacity that could manifest in an alteration in their ability to translate environmental events into long-term changes in gene expression. This past year we have pursued both the mechanism underlying the altered expression of the ESC/E(Z) pathway and the effects of ovarian steroid exposures on gene expression in PMDD versus controls. First, preliminary evidence from a whole small RNA sequencing (targeting micro-RNA expression) shows a significantly increased expression of several micro-RNAs in LCLs from women with PMDD compared to those in controls. Additionally, several of these differentially expressed micro-RNAs target the ESC/E(Z) pathway, and, therefore, could contribute to the observed dissociation between increased expression of the ESC/E(Z) pathway genes and decreased protein levels of this same pathway in PMDD. Second, we employed two stock neuronal cell lines: Luhmes Cells, which are immortalized neuronal precursor cell lines (both undifferentiated/neuronal precursors and differentiated/mature dopaminergic neurons), as well as SH-SY5Y an immortalized neuroblastoid cell line characterized by an adrenergic and dopaminergic phenotype. Both stock neuronal cell lines contained evidence of ESC/E(Z) gene complex function and responsivity to ovarian steroids. Thus, these data provide strong supportive evidence of the neural relevance of our initial findings in lymphoblastoid cell lines (immune-origin tissue) in several more pathophysiologically-relevant tissues (i.e., human NPCs, two stock neuronal cell lines). Finally, after estradiol exposure we recently identified abnormalities of intra-cellular calcium regulation and the endoplasmic reticulum stress response. These latter findings of altered intracellular calcium signaling after estradiol exposure will be followed up with calcium imaging; however, they also could provide a path towards developing novel therapeutics for this condition. In a parallel series of functional genomic studies employing LCLs and h-IPSCs from women with PPD and in a separate group of women with first-onset postpartum psychosis (PPP) we have completed whole transcriptome sequencing. Whole transcriptome RNA-sequencing is completed in LCLs from controls and women with PPD (n=9 in each group) under conditions of untreated baseline, supraphysiologic (high) combined estradiol and progesterone (replicating pregnancy), and hormone withdrawal (replicating hormonal events of parturition). Preliminary findings revealed multiple differentially expressed genes in all conditions meeting FDR corrections, particularly in the high E2+P conditions between cases and controls, including a translational regulator that ensures constant high levels of translation upon a variety of stress conditions, which was significantly decreased in cases. Neural progenitor cells have been created from women with PPP and controls and they are currently being sequenced and analyzed via RNA-seq.